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Phospho-ERK (Thr202/Tyr204) Cellular Assay Kit

HTRF® cellular assay kit for measuring phosphorylated ERK1/2 directly in cells

Based on our homogeneous and robust HTRF assays, Cellul'erk is designed for detecting and studying activated Erk1/2 directly in whole cells. Using streamlined protocols, amenable to low-volume format, this kit can be used from basic research to High Throughput drug screening.

Features

  • Simple assay protocols: an alternative to Western Blot or ELISA
  • Homogeneous, direct detection
  • HTRF robustness, sensitivity and reliability
  • Suitable for exploring endogeneous and over-expressed receptors
  • Use in many types of cells, including primary
  • All inclusive: all reagents in one kit

Applications

  • Gi, Gs, or Gq coupled receptor activation
  • Mitogen-activated protein kinase (MAPK) pathway (RTK activity)
  • β -Arrestin signaling
  • Biotherapeutics: mechanism of action

Assay can be run with cell lysates or using whole cells.  ERK is activated and after lysis of the cell membrane, the phosphorylated ERK released can be detected using the HTRF kit reagents via a sandwich assay format using 2 different specific monoclonal antibodies: the anti-phospho-ERK antibody labeled with d2 and the anti-ERK antibody labeled with Eu3+-cryptate. These antibodies may be pre-mixed and added in a single dispensing step to further streamline the protocol.

Cellulerk assay theory

The assay can be run under a two-plate protocol. It can also be further streamlined to a one-step assay.

Two-plate assay protocol:

Cells are plated, stimulated and next lysed in the same 96-well culture plate. Lysates are then transferred to the assay plate for the detection of phosphorylated ERK by HTRF reagents. This protocol allows cell viability and confluence to be monitored.

Cellulerk two plate protocol

One-plate assay protocol:

Detection of phosphorylated ERK with HTRF reagents is performed in a single plate used for plating, stimulation and lysis. No washing steps are required. This protocol, HTS designed, allows miniaturization while maintaining HTRF quality.

Cellulerk one plate protocol

 

Cellulerk pathway

GPCRs act via G proteins to regulate a wide range of cellular functions. Upon stimulation, these receptors activate effectors like adenylate cyclase and phospholipase C which influence not only intracellular concentrations of second messengers (cyclic AMP, diacylglycerol, inositol 1,4,5 trisphosphate and Ca2+), but also mediate Extracellular signal Regulated Kinase (ERK1/2) phosphorylation. Activation of Gai/o, Gas, Gaq/11 or Ga12/13 modulates ERK1/2 activation via numerous mechanisms. In addition, both a and ß? subunits of G proteins can stimulate ERK1/2 phosphorylation through transactivation of receptor tyrosine kinases (RTKs). GPCRs have also been shown to mediate ERK1/2 activation in a G protein-independent but ß-arrestin dependent manner.

1. Western Blot versus HTRF assay

A431 cells were grown in a T175 flask 37°C, 5%Co2, 2days. Stimulation was done with 100nM EGF for 10min. After elimination of cell culture medium, 3ml of supplemented lysis buffer was added and incubated for 45min. Soluble supernatants were collected after 10min centrifuging.

Total protein concentration was determined by BCA assay: 20.106 cells lysed under 3ml corresponds to 3.2mg/ml of total proteins. (50.000 cells per well corresponds to 25µg of total proteins) 16µl of serial lysates dilutions were dispensed and analysed side-by-side by WB and HTRF assay.

The HTRF assay is 8-fold more sensitive than the western-blot : 200 cells can be detected by using Cellul'erk while 1960 cells are needed for the Western-blot.

Cellulerk western blot graph

 

2. Gaq/i coupled receptor activation

Results obtained on CHO-CCR5 (25,000 cells) activated with Rantes & MIP-Iß for 10', using the two-plate assay protocol.

Gaq/i coupled receptor activation.

Cellulerk rgqi coupled graph

 

3. RTK activation: functional characterization of a cell line

Results obtained on cells expressing EGFR1, pre-treated or not with Cetuximab (therapeutic monoclonal antibody) or erlotinib (Tyrosine kinase inhibitor) for 2 hours at 37°C. Cells were then stimulated for 10 min with EGF.

The results show that the 2 compounds can efficiently inhibit ERK phosphorylation. They confirm that the monoclonal therapeutic antibody Cetuximab targets the EGFR1 binding site, and therefore prevents the activation of the ERK signaling pathway.

Cellulerk phospho erk inhibition graph

 

4. Screening robustness:

CHO-M1 (5,000 cells/well - 384-well small volume plate) were stimulated with Carbachol for 10 minutes at RT.

Z' values in fifty replicates were calculated between unstimulated cells (basal level) and 2 selected Carbachol concentrations,1.3 µM and 4 µM, corresponding to the EC80and EC100 respectively.

Assay was performed using the One-plate assay protocol on a robotic system from CyBio™ (CyBi®vario equipped with 384/25µL pipeting head). Results were read on PheraStarplus (BMGLabtech).

Cellulerk screening robustness graph

Ordering Info

DescriptionCat. noProduct insertMSDS
Phospho-ERK - 500 tests64ERKPEG
Phospho-ERK - 10,000 tests64ERKPEH
Phospho-ERK - 50,000 tests64ERKPEI

Companion products

DescriptionCat. noProduct insertMSDS
Phospho-ERK control lysate62ERKTDA
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Cellular kinase blocking reagent - 2 ml64KB1AAC
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Cellular kinase blocking reagent - 6 ml64KB1AAD
-
Cellular kinase lysis buffer #1 - 130 mL64KL1FDF
-
Total ERK1/2 Assay kit - 500 tests64NRKPEG
-
Total ERK1/2 Assay kit - 10,000 tests64NRKPEH
-
Total ERK1/2 Assay kit - 50,000 tests64NRKPEI
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